The control of gene expression is central to the growth and development of all organisms. Disrupting the normal control mechanisms can lead to cell death or transformation. In other cases, subverting the cells transcription apparatus can form the basis for viral infection. Appropriately, to understand the mechanisms of eukaryotic transcription is a major goal in modern biology. This is a proposal to study transcription of RNA polymerase II genes from the model organism Acanthamoeba castellanii. The long-term goal is to establish principles of eukaryotic gene expression at the molecular level. The in vivo structure of an actin gene from Acanthamoeba castellanii will be determined. The results will identify promoter regions that interact with protein in vivo and will be used as a reference point for in vitro studies. An in vitro transcription system will be developed form Acanthamoeba castellanii using the Acanthamoeba actin I and myosin II genes. Promoter elements required for accurate transcription in vitro from the actin gene will be identified and the number and type of factors required for transcription will be determined. The interaction between factors, RNA polymerase II and DNA will be examined by footprinting. The steps of preinitiation complex assembly that lead to initiation will be defined in terms of polymerase and factor binding as well as DNA unwinding or other conformational changes. Purification of transcription factors will be initiated using a combination of conventional and promoter affinity methods.